Figure S1 A BC D E HNRNPUL1 GFP Kasumi-7_HA-tag_clones 30 stop HA-tag 2A Kasumi-7 parent 20 125 100 RT + + + - + + + - 190 10 125 80 * fold-increase * 1.5 100 58 1.0 80 (kb) 0 58 (kDa) 46 anti-HA blot 32 MEF2D-GFP HNRNPUL1(not shared (kDa) with MEF2D-HNRNPUL1 ) -GFP F G KOPN46 KOPN61 KOPN70 KOPN71 YAMN96 KOS20 P30/OHK l5 Igm cell number pre-BCR staining with the antibodies staining with isotype control H JALSG_ALL202-U TARGET Figure S1. Generation of genome-edited Kasumi-7 cells, characterization of MEF2D-fusion-bound regions, analysis of pre-BCR expression in MEF2D-ALL cells, and analysis of pre-BCR-associated gene expression in clinical samples (A) Schematic drawing of the 3’-end of the MEF2D-HNRNPUL1 fusion gene coding sequence in genome-edited Kasumi-7 (K7-HA-GFP) cells (upper panel). The 3’-end HA- tag enables identification of the fusion protein using an anti-HA antibody (lower panel). GFP, which is expressed from the 2A sequence, enables fluorescent identification of the tagged cells. (B) Representative clones generated by genome-editing. Three independent clones were subjected to a western blot analysis with an HA-specific antibody. In Clone 1, both the MEF2D-HNRNPUL1 fusion (arrowhead) and non-rearranged HNRNPUL1 allele (arrows) were HA-tagged. In Clone 2, only the MEF2D-HNRNPUL1 fusion was HA-tagged. In clone 3, only the non-rearranged HNRNPUL1 allele was HA-tagged. Clone 2 was used throughout the remainder of the study. *: Non-specific bands. (C) RT-PCR analysis demonstrating that in clone 2 from B, the MEF2D-HNRNPUL1 fusion was HA-tagged, whereas the non-rearranged HNRNPUL1 allele was not tagged. Kasumi-7 parent, bulk genome-edited, and K7-HA-GFP clone 2 cells were subjected to an RT-PCR analysis to determine the expression of transcripts encoding MEF2D and GFP, as well as HNRNPUL1 and GFP. For the latter, primer sets designed to amplify the transcript encompassing non-rearranged HNRNPUL1 and GFP were used. (D) Proportion of genomic regions occupied by the MEF2D-HNRNPUL1 fusion protein in K7-HA-GFP cells (upper panel). Schematic drawing of the most enriched motif in the MEF2D-HNRNPUL1-bound regions (lower panel). (E) ChIP-qPCR analysis of MEF2D-HNRNPUL1 occupancy in the genomic regions near the transcription start sites of the indicated genes in K7-HA-GFP cells. The fold- increases in DNA precipitated by an anti-HA antibody were compared with the DNA precipitated by normal IgG and are shown as means ± standard deviations (n=3). (F) KEGG B cell receptor signaling pathway. Genes assigned to super-enhancers associated with MEF2D-HNRNPUL1 occupancy in K7-HA-GFP cells are shown in red. (G) Pre-BCR expression in the indicated MEF2D-ALL cell lines was detected using dual-staining for Igm and l5 (upper panel) and single-staining for the pre-BCR complex (lower panel). (H) Expression of genes associated with pre-BCR-positive versus -negative BCP-ALL in two clinical cohorts. MEF2D-ALL and non-MEF2D-ALL samples were compared (PBX1-rearranged samples were excluded), and the x-axis and y-axis represent the mean expression levels and log2-fold-change values (MEF2D-ALL/non-MEF2D-ALL) calculated using DESeq2, respectively. Figure S2 AdCas-KRAB (BFP) B Ctr_1 Ctr_2 guide- Ctr_3 RNA(GFP) GFP-pos. IGLL1_1 GFP-neg. BFP-neg. BFP-pos. IGLL1_2 IGLL1_3 VPREB1 Neg. Ctr. for value) (relativeGFP % l5 staining Crispr-I culture period (days) guide-RNA(GFP) guide-RNA(GFP) Ctr._1 cell number cell number Crispr-I Ctr._2 Crispr-I Ctr._3 Crispr-I IGLL1_1 Crispr-I IGLL1_2 Crispr-I IGLL1_3 Crispr-I VPREB1 l5 Figure S2. Crispr-I-mediated hindrance of enhances, involving MEF2D-HNRNPUL1-bound regions near TSSs of IGLL1 and VPREB1 genes, reduces pre-BCR expression and attenuates Kasumi-7 cell growth. (A) Kasumi-7 cells were infected with lentiviruses engineered to co-express dCas-KRAB and BFP, as well as lentiviruses engineered to co-express guide-RNA and GFP. The cells were then subjected to flow cytometry to determine the expression of BFP, GFP, and pre-BCR. Pre-BCR expression was not altered in BFP-negative cell fractions, regardless of GFP expression. In BFP-positive cell fractions, pre-BCR expression was attenuated only in GFP-positive cells infected with the guide-RNA lentivirus that targeted enhancers of IGLL1 and VPREB1. Infection with a lentivirus encoding control guide RNA had no effects. (B) The %GFP values in BFP+ cells are presented as relative values over the indicated culture period. Impedance of the IGLL1 and VPREB1 enhancers led to impaired cell growth. Figure S3 A B KOPN46KOPN61 KOPN70 KOPN71 KOS20 YAMN96 P30/OHK KOPN46 Ibr. Ibr. Ibr. Ibr. Ibr. Ibr. Ibr. vehicle vehicle vehicle vehicle vehicle vehicle vehicle Dasa. Dasa. Dasa. Dasa. Dasa. Dasa. Dasa. PRT. PRT. PRT. PRT. PRT. PRT. PRT. 135 135 100 100 100 80 80 80 KOPN61 58 MEF2D-fusions 58 58 46 KOPN70 46 46 GAPDH KOPN71 C Kasumi-7Kasumi-9 TS-2 D cell number KOS20 FK506 - + - + - + Kasumi-7 MEF2D-fusions p30/OHK vehicle Dasa. Kasumi-9 PRT062607 GAPDH YAMN96 Ibr. Neg. Ctr. pre-BCR TS-2 l5 Igm FK506-treated vehicle Figure S3. Effects of drug on the expression of MEF2D-fusion protein and pre-BCR (A) Expression of the MEF2D-fusion protein in the indicated MEF2D-ALL cell lines after treatment with dasatinib, PRT062607, and ibrutinib as in Figure 3F. (B) Expression of pre-BCR on the indicated MEF2D-ALL cell lines after treatment with dasatinib, PRT062607, and ibrutinib as in Figure 3G. (C) Insensitivity of MEF2D-fusion protein expression to FK506 treatment (100 nM, 3 days) in MEF2D-ALL Kasumi-7, Kasumi-9, and TS-2 cells. (D) Insensitivity of pre-BCR expression to FK506 treatment (100 nM, 3 days) in Kasumi-7, Kasumi-9, and TS-2 cells. Figure S4 5 kb CD79A 5 kb 0.2 IGLL1 0.2 VPREB1 0.2 ATAT-seq 5 kb 0 0 0 MEF2D-fusion 0.7 0.9 0.4 A B 0 0 0 8 6 21 SREBF1 0 0 0 10 10 14 FOS 0 0 0 EGR1 0.3 0.3 0.4 EGR1 0 0 0 BCL6 2 0.9 0.9 0.4 FOS BCL6 0 0 0 IRF8 13 13 8 RUNX1 0 0 0 ERG 0.5 0.5 0.5 RUNX1 0 ERG 0 0 0 NFATC1 0.15 0.15 0.5 NFATC1 0 0 0 SREBF1 6 4 16 CREB1 SATB1 -2 0 0 0 TCF12 H3K27ac 1.6 1.6 1.6 IKZF1 SE 0 0 0 YY1 ARID5B -4 PPP3CC PIK3CD PLCG2 TCF3 20 kb IRF4 ATAT-seq 0.2 0.1 100 kb 0.21 0 0 0 50 kb SOX4 MEF2D-fusion 0.2 0.5 0.8 0 0 0 MEF2A 15 18 19 SREBF1 HIVEP2 0 0 0 EBF1 14 22 15 FOS 0 0 0 MEF2D 0.4 0.8 0.4 EGR1 0 0 0 PAX5 0.2 0.2 0.5 IRF2 BCL6 0 0 0 KLF13 1.5 1.5 3 RUNX1 0 0 0 ELF1 1.5 0.6 1.2 ETS1 ERG 0 0 0 LEF1 0.3 0.25 0.25 NFATC1 0 0 0 MYB 20 20 9 CREB1 IRF1 0 0 0 presence H3K27ac 0.8 0.7 0.9 absence SE 0 0 0 C FOS 5 kb MEF2D 20 kb SREBF1 20 kb EGR1 1 0.15 10 kb 0.15 0.35 ATAT-seq 0 ATAT-seq 02 0 0.5 1 00.35 MEF2D-fusion 0 0 0 0 MEF2D-fusion 30 100 100 50 SREBF1 0 0 SREBF1 0 50 0 50 22 131 FOS 0 0 0 0 FOS 2 0.6 0.4 3.4 EGR1 0 0 0 0 EGR1 0.5 0.25 0.25 0.27 BCL6 0 0 0 0 BCL6 8 0.25 2.5 1.2 RUNX1 0 0 0 0 RUNX1 1 1.25 1.5 1.4 ERG 0 0 0 0 ERG 0.3 0.3 0.3 0.5 NFATC1 0 0 0 0 NFATC1 300 57 150 90 CREB1 0 0 CREB1 0 0 1.2 H3K27ac 2 1 1.1 H3K27ac 0 0 0 0 SE SE NFATC1 BCL6 ERG 20 kb RUNX1 100 kb 0.15 0.2 0.12 100 kb 0.15 50 kb ATAT-seq 0 0 1.2 ATAT-seq 0.5 00.9 00.7 MEF2D-fusion 0 0 0 0 MEF2D-fusion 157 16 20 16 SREBF1 0 0 0 0 SREBF1 20 15 6 13 FOS 0 0 0 0 FOS 0.6 0.5 0.3 0.6 EGR1 0 0 0 0 EGR1 3.5 0.35 0.5 0.3 BCL6 0 0 0 0 BCL6 3 14 4 4 RUNX1 0 0 0 0 RUNX1 0.8 0.9 0.5 1 ERG 0 0 0 0 ERG 0.3 0.23 0.2 0.35 NFATC1 0 0 0 0 NFATC1 32 10 5 73 CREB1 CREB1 0 0 0 0 H3K27ac 1 0.8 1.2 1.2 H3K27ac 0 0 0 0 SE SE IRF8 10 kb ATAT-seq 0.12 00.25 MEF2D-fusion 0 1 25 D E SREBF1 0 100 FOS 0 0.7 EGR1 0 0.4 BCL6 0 1.5 RUNX1 0 0.9 ERG HA HA 0.5 0 1 0.90 0.83 NFATC1 0.5 HA 0 1 0.92 0.83 CREB1 50 1 1.1 0.80 0 ERG RUNX1 H3K27ac 0.7 SE 0 1 0.58 0.45 NFATC1 1 0.40 0.23 GAPDH 1 0.47 0.27 GAPDH GAPDH number Cell (relative to control) 0 Figure S4.